We are developing the essential components of a system with which to investigate the pathogenesis of Lyme disease and the basic biology of the causative agent, Borrelia burgdorferi. Sensitive and accurate detection of organisms is necessary in order to compare their presence (or absence) with disease symptoms and to facilitate an accurate diagnosis of infection. Characterization of key structural and regulatory elements of an organism is requisite to identifying potential pathogenic mechanisms. Ultimately, critical proteins can be genetically manipulated and reintroduced and the outcome on infection and disease assessed. 1. Detection: A sensitive and specific PCR assay has been developed from a chromosomally encoded B. burgdorferi DNA sequence. Sequence comparisons permit the design of broadly reactive but burgdorferi-specific primers. A modified protocol using 'nested' primers has increased the sensitivity and specificity in the presence of excess eukaryotic DNA. When tested against 31 different B. burgdorferi isolates of broad geographic and biological origins, all were detected. 2. Molecular Characterization: Several features of B. burgdorferi are being addressed. Classification of isolates by reactivity with different sets of PCR primers subdivides burgdorferi strains into 2 groups. Analysis and sequencing of the genes encoding 2 major outer surface proteins has identified potential mechanisms for variation in expression of these immunodominant components. Modification of a plating protocol has provided a more rapid and efficient means of cloning organisms and has permitted the clonal analysis of variants. Isolating and studying genes whose expression is increased after a temperature upshift (heat shock genes) will provide information about their roles in cell growth, pathogenesis, plasmid replication and adaptation to stress. 3. Gene Transfer: In order to allow genetic analysis of B. burgdorferi, two methods for gene transfer into Borrelia are being developed. Vectors have been constructed to allow transferred DNA to integrate or transpose into the chromosome. Parameters for transformation using electroporation are being tested. Conditions for the alternative to electroporation, mating with E. coli, are being established.